This invention relates to 5,11-dioxa-benzo[b]fluoren-10-one and 5-oxa-11-thia-benzo[b]fluoren-10-ones which are useful as estrogenic agents.
The pleiotropic effects of estrogens in mammalian tissues have been well documented. (Dey, M.; Lyttle, C. R.; Pickar, J. H. Maturitas (2000), 34(S2), S25-S33. Speroff, L Ann. N. Y. Acad. Sci. (2000), 900, 26-39. Nozaki, M. Ernst Schering Res. Found. Workshop (2000), Suppl. 4, 115-125). The estrogen receptor (ER), a member of the nuclear hormone receptor family, regulates transcription through its interactions with a large number of proteins including co-activators and co-repressors (collectively referred to as coregulators) and an estrogen response element (ERE). In addition to its ability to effect the cellular transcription machinery through the ERE, ER can also affect transcriptional processes independent of its direct interaction with DNA. For example, it has been demonstrated that 17xcex2-estradiol can inhibit IL-6 promoter activity. This inhibition requires 17xcex2-estradiol binding to the receptor, but does not depend on having a functional DNA-binding domain (Ray, A.; Prefontaine, K. E.; Ray, P. J. J. Biol. Chem., 1994, 269, 12940). Even the unliganded receptor may affect the transcription process after phosphorylation of serine residues, especially in the AF-1 containing AB domains of the receptor.
Recently, a second ER receptor (ERxcex2) with high affinity for 17xcex2-estradiol has been identified. A comparison of the physical structure of ERxcex2 with the first to be identified estrogen receptor (ERxcex1) reveals that ERxcex2 is shorter in length (530 AA vs. 595 AA) but contains the same functional domains although the AB domains of ERxcex2 are somewhat truncated relative to ERxcex1 (148AA vs. 180AA) and not surprisingly, the AF-1 activation potential between the two receptors is different (McInerney, Eileen M.; Weis, Karen E.; Sun, Jun; Mosselman, Sietse; Katzenellenbogen, Benita S. Endocrinology (1998), 139(11), 4513-4522). The C domain (DNA-binding domain) displays remarkable homology between the two receptors (96%) and a fortiori, the two receptors would be expected to bind with similar affinities to a given ERE. However, although it has been shown that the two receptors bind to ERE""s vitogenellin, c-fos, c-jun, pS2, cathepsin D, and acetylcholine transferase, they do not necessarily bind with the same affinity (Hyder, S. M., Chiappefta, C., Stancel, G. M. Biochem. Pharmacol. (1999), 57, 597-601). In contrast, the E domain (ligand binding domain or LBD) of the two receptors share only a 60% homology. However, structural analyses of the two receptors indicates that the residues in the ligand contact area are very similar, with only two residues different (ERxcex1 421(Met) ERxcex2 373(Ile); ERxcex1 384 (Leu) ERxcex2 336(Met)). Additionally, the variations in the overall sequence of the two receptors may also lead to different interactions between the subtypes and the various coregulatory proteins that enable or modify the ER transcriptional machinery. In fact, preliminary studies suggest that the coregulator SRC-3 interacts to a much greater extent with ERxcex1 than with ERxcex2 (Suen, Chen-Shian; Berrodin, Thomas J.; Mastroeni, Robert; Cheskis, Boris J.; Lyttle, C. Richard; Frail, Donald J. Biol. Chem. (1998), 273(42), 27645-27653).
Besides the differential interaction of the two receptors with various coregulatory proteins, the two receptors also have tissue distribution that is not coextensive. Even within a given tissue where both receptors are coexpressed there is sometimes localization of one of the receptors in a given cell-type. For example, in the human ovary, both ERxcex1 and ERxcex2 RNA expression can be detected. Immunostaining demonstrates that ERxcex2 is present in multiple cell types including granulosa cells in small, medium and large follicles, theca and corpora lutea, whereas ERxcex1 was weakly expressed in the nuclei of granulosa cells, but not in the theca nor in the corpora lutea (Taylor, A. H.; Al-Azzawi, F. J. Mol. Endocrinol. (2000), 24(1), 145-155). In the endometrium, immunostaining showed both ERxcex1 and ERxcex2 in luminal epithelial cells and in the nuclei of stromal cells but, significantly, ERxcex2 appears to be weak or absent from endometrial glandular epithelia (Taylor, et al). Epithelial cells in most male tissues including the prostate, the urothelium and muscle layers of the bladder, and Sertoli cells in the testis, are also immunopositive for ERxcex2. Significant ERxcex2 immunoreactivity has been detected in most areas of the brain, with the exception of the hippocampus, a tissue that stained positive for only ERxcex1, ibid.
Estrogens have been shown to exert a positive effect on the cardiovascular system that may help to explain the increased risk of cardiovascular disease observed in the post-menopause period. While some of the cardiovascular benefit may occur through estrogen action on the liver via upregulation of the LDL receptor (thus decreasing LDL levels, presumably an ERxcex1 mediated response), it is also likely that direct action on the arterial wall also has a role. It has been demonstrated that after a vascular injury event (denudation of rat artery), ERxcex2 message in the endothelial cells is upregulated by as much as 40 times that of ERxcex1 (Makela, Sari; Savolainen, Hanna; Aavik, Einari; Myllarniemi, Marjukka; Strauss, Leena; Taskinen, Eero; Gustafsson, Jan-Ake; Hayry, Pekka. (1999), 96(12), 7077-7082). In addition, 17xcex2-estradiol was able to inhibit the vascular injury response in an ERxcex1 knockout mouse, although this same response was also inhibited in an ERxcex2 knockout mouse (Iafrati, Mark D.; Karas, Richard H.; Aronovitz, Mark; Kim, Sung; Sullivan, Theodore R., Jr.; Lubahn, Dennis B.; O""Donnell, Thomas F., Jr.; Korach, Kenneth S.; Mendelsohn, Michael E. Nat. Med. (N.Y.) (1997), 3(5), 545-548. Karas, Richard H.; Hodgin, Jeffrey B.; Kwoun, Moon; Krege, John H.; Aronovitz, Mark; Mackey, William; Gustafsson, Jan Ake; Korach, Kenneth S.; Smithies, Oliver; Mendelsohn, Michael E. Proc. Natl. Acad. Sci. U.S.A. (1999), 96(26), 15133-15136). Provided that the response is not being inhibited by a yet unidentified estrogen receptor, it is likely that the injury response could be inhibited by ligands that are selective for either one of the two receptors.
When the typical estrogen binds with an ER receptor, the receptor dissociates from HSP 90 as well as other molecular chaperones, and dimerizes with another receptor. Since this mechanism of activation is shared by both ER receptors, the possibility exists for heterodimerization to take place in tissues where both receptors are expressed. Indeed, heterodimers of ERxcex1 and ERxcex2 bind DNA with an affinity equal to that of ERxcex1 homodimers and greater than ERxcex2 homodimers (Cowley, Shaun M.; Hoare, Susan; Mosselman, Sietse; Parker, Malcolm G. J. Biol. Chem. (1997), 272(32), 19858-19862).